A pore-forming toxin produced by Aeromonas sobria activates Ca2+ dependent Cl- secretion

Bacteria produce many types of hemolysin that induce diarrhea by mechanisms that are not completely understood. Aeromonas sobria hemolysin (ASH) is a major virulence factor produced by A. sobria, a human pathogen that causes diarrhea. Since epithelial cells in the intestine are the primary targets of hemolysin, we investigated the effects of ASH on ion transport in human colonic epithelial (Caco-2) cells. ASH increased short-circuit currents (Isc) in a dose-dependent manner, and it also activated a 125I efflux from Caco-2 cells. ASH-induced Isc increases and 125I efflux activations were both suppressed by low Ca2+ levels in the extracellular solution or by pretreatment with the Ca2+ chlelator BAPTA-AM. Intracellular Ca2+ levels were increased by ASH in a biphasic fashion characterized by a rapid sharp increase (peak 1) followed by a sustained low plateau (peak 2). ASH-induced peak 1 was inhibited by pretreatment with pertussis toxin, indicating that Ca2+ was mobilized from intracellular stores, and peak 2 was induced by an influx of extracellular Ca2+. Peak 2 but not peak 1 was related to Cl- secretion. These results indicate that ASH activates Ca2+-dependent Cl- secretion.     

A method for simultaneous recording of electrical activities and spectrophotometric signals from brain slice preparations in vitro

Organ spectrophotometry has been applied to analyze cytochrome redox changes in brain slice preparations. An interface-chamber method for maintaining metabolism of brain slice tissues was devised to reduce noise on recording traces of spectrophotometric signals, and then used for continuous monitoring and simultaneous recording of electrical and optical signals from brain slices. With this method, the noise level during the recording of redox states of cytochromes was decreased to 0.0004 A unit.     

Functional interleukin 2 receptors on B cells lacking Tac antigens

Human B lymphoblastoid line, SKW 6-4, cells were induced to IgM-secreting cells by high concentrations of interleukin 2 (IL 2). These cells were found to be unreactive with anti-Tac antibody and did not express mRNA detectable for Tac antigen. In Scatchard plot analysis, low-affinity IL 2-binding sites were found on SKW 6-4 cells. Moreover, analysis of the IL 2-binding molecules revealed ones (molecular weight 70,000 and 75,000) distinct from Tac antigen. It is conceivable that IL 2 exerts its effect through its interaction with these novel IL 2-binding molecules in SKW 6-4 cells.     

Continuous administration of gonadotrophin-releasing hormone agonist during the luteal phase in IVF

BACKGROUND: It has been reported that ceasing the administration of gonadotrophin-releasing hormone (GnRH) agonist causes a profound suppression of circulating serum gonadotrophins. A comparative prospective and randomized study was conducted to investigate the effect of continuous administration of GnRH agonist during the luteal phase in an ovarian stimulation programme for IVF. METHODS: GnRH agonist was administered intranasally from the midluteal phase of the previous cycle, and pure FSH administration started on cycle day 7. In the continuous-long protocol (cL) group (n = 161 ), GnRH agonist administration was continued until 14 days after oocyte retrieval. In the long protocol (L) group (n = 158 ), GnRH agonist was administered until the day before human chorionic gonadotrophin (HCG) administration. RESULTS: The implantation rate and live birth rate per unit of transferred embryos were significantly higher in the cL group than the L group (P < 0.05 ). Serum LH and FSH concentrations on the day of, and 1 day after, HCG administration were significantly lower in the L group than the cL group (P < 0.01 ). CONCLUSIONS: Continuation of GnRH agonist administration during the luteal phase might facilitate implantation, and prevent the profound suppression of serum gonadotrophins.     

Characterization of autoantigens relevant to experimental autoimmune orchitis (EAO) in mice immunized with a mixture of syngeneic testis homogenate and Klebsiella O3 lipopolysaccharide

Experimental autoimmune orchitis (EAO) in mice has been induced by repeated injection of a mixture of syngeneic testis homogenate and Klebsiella O3 lipopolysaccharide (KO3 LPS) as a potent adjuvant. The antisera obtained from mice with EAO lesions defined several antigens with apparent molecular weights (MW) of 38,000 (38 kd), 86 kd, 100 kd, and greater than 200 kd by the immunoblotting method. These antigens were organ-specific and exclusively present on the acrosome of spermatozoa, suggesting that these acrosomal antigens were highly relevant to EAO. It was found that the antigen with a fairly high MW (greater than 200 kd) was expressed on spermatozoa from the epididymis. Furthermore, the acrosomal 86 kd antigen was predominantly expressed in the testis, while the 100 kd antigen was dominant in the spermatozoa from the epididymis. It was therefore suggested that the 86 kd and 100 kd antigens in the acrosome were differentially expressed on the process of maturation of spermatozoa.     

A new pseudo-peptide of Arg-Gly-Asp (RGD) with inhibitory effect on tumor metastasis and enzymatic degradation of extracellular matrix

A series of pseudo-peptide analogs of the Arg-Gly-Asp (RGD) sequence of fibronectin have been synthe-sized, and their anti-metastatic effects in mice and inhibitory effects on tumor cell invasion in vitro have been examined. The partially modified retro pseudo-peptide of RGD, R_rev-COCH₂CO-D (FC-63), was more effective in inhibiting tumor metastasis than the original RGDS peptide. Replacement of the malonyl moiety of FC-63 with a carboxyethylene linkage (R_rev-COCH₂CH₂-D, FC-303 ) achieved more potent inhibition of lung metastasis of melanoma cells than FC-63. Among the analogs, FC-336, a p-xylylendiamine derivative having two FC-303 moieties, showed the most potent inhibitory effect on experimental lung metastasis produced by i.v. co-injection with B16-BL6 melanoma or colon 26 M3.1 cells in a dose-dependent manner. Multiple administrations of FC-336 after tumor inoculation also showed efficient therapeutic potency against spontaneous lung metastasis of B16-BL6 melanoma in mice. Furthermore, FC-336 effectively inhibited the invasion, migration and adhesion of tumor cells in vitro, but its inhibitory effects were not more than those of RGDS peptide. Zymography analysis revealed that FC-336 inhibited the degradation of gelatin substrate by matrix metalloproteinases (MMPs) produced by tumor cells, while the RGDS peptide did not affect the enzymatic degradation. These findings indicate that the pseudo-peptides of the RGD sequence, possessing the inhibitory property of the degradation by MMPs differently from original RGD-containing peptides, may be advantageous and useful in preventing tumor metastasis. © Rapid Science 1998     

Molecular cloning of cDNAs expressing SS-B/La protein

Using serum from a patient with Sj?gren's syndrome containing a high titer of anti-SS-B/La antibody, cDNA clones (a representative clone was called pA158) were isolated from a human fibroblast cDNA library in lambda gt11 expression vector. After subcloning of pA158 cDNA into an expression plasmid vector pEX-2, a large amount of the recombinant fusion protein with cro-beta-galactosidase (called pA158EX) was obtained in E. coli culture containing the recombinant pEX-2. Antibodies against pA158EX were purified from the patient serum by Sepharose 4B conjugated with the purified pA158EX protein. Immunofluorescent staining of HEp-2 cells with the anti-pA158EX antibodies showed a speckled nuclear staining. In immunoblot analysis, the anti-pA158EX antibodies reacted with 50 kDa protein that was compatible with SS-B/La protein. Immunoprecipitation of leukocyte lysate with the anti-pA158EX antibodies and the following RNA analysis showed that the antibody precipitated Y5 RNA. These findings indicate pA158 is a cDNA for SS-B/La protein. The purified fusion protein was used for enzyme-linked immunosorbent assay (ELISA). Optical density values of anti-SS-B positive sera were high, but those of anti-SS-B negative sera and healthy donor sera were low. In the Northern blot using human RNA and pA158 cDNA, a single band about 1.8 kb was recognized. A full-length cDNA was further obtained by screening of pcD library using pA158 cDNA as a probe.     

Preparation of Asialofetuin-Labeled Liposomes with Encapsulated Human Interferon-γ and Their Uptake by Isolated Rat Hepatocytes

The selective delivery of human recombinant interferon (IFN)- to isolated rat hepatocytes was studied with asialofetuin (AF)-labeled liposomes. AF-liposomes containing buffer solution were initially prepared by the detergent removal method, and IFN- was subsequently encapsulated by the freeze-thawing method without loss of activity. Virtually no free [³²P]IFN- was internalized into isolated rat hepatocytes, whereas AF-liposomes containing [³²P]IFN- were taken up to a significant degree. Liposomal binding to the hepatocytes (estimated at 4°C) was one-fifth of the uptake (estimated at 37°C). Since the uptake was inhibited by the addition of free AF, AF-liposomes may be taken up by the action of galactose-binding protein on the hepatocytic cell surface. The liposome preparation method reported in this paper provides a useful means for the encapsulation of unstable macromolecules into AF-liposomes. AF-liposomes were found effectively to carry IFN- into hepatocytes in vitro.     

Risk factors for hepatocellular carcinoma at baseline and 1 year after initiation of nucleos(t)ide analog therapy for chronic hepatitis B

Nucleos(t)ide analogs (NAs) cannot completely suppress the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). This study aimed to identify the risk factors for HCC development in naïve CHB patients treated with current NA. Patients receiving NA (n = 905) were recruited retrospectively from the 17 hospitals of the Japanese Red Cross Liver Study Group. All treatment-naïve patients had been receiving current NA continuously for more than 1 year until the end of the follow-up. We analyzed the accuracy of predictive risk score using the area under receiver operating characteristic curve. The albumin–bilirubin (ALBI) score was significantly improved by NA therapy (−0.171 ± 0.396; p < 0.001 at Week 48). A total of 72 (8.0%) patients developed HCC over a median follow-up of 6.2 (1.03–15.7) years. An independent predictive factor of HCC development was older age, cirrhosis, lower platelet counts at baseline and ALBI score, and alpha-fetoprotein (AFP) at 1 year after NA therapy according to multivariate analysis. The accuracy was assessed using the PAGE-B, mPAGE-B, aMAP, APA-B, and REAL-B scores that included these factors. Discrimination was generally acceptable for these models. aMAP and REAL-B demonstrated high discrimination with 0.866/0.862 and 0.833/0.859 for 3- and 5-year prediction from the status of 1 year after NA therapy, respectively. Baseline age and platelet count, as well as ALBI and AFP one year after NA, were useful for stratifying carcinogenesis risk. The aMAP and REAL-B scores were validated with high accuracy in Japanese CHB patients.